Technical Field of the Invention
The present invention relates to reconstructed skin having a constitutive pigmentation, and to the use thereof in methods for evaluating skin pigmentation phenomena; it thus relates to methods for evaluating agents capable of modulating this pigmentation, whether it is constitutive or induced. It also relates to a method for preparing such skin models.
Description of Background and/or Related and/or Prior Art
Skin color is principally due to the presence of a pigment, melanin, in the epidermis. Melanin is synthesized by specific dendritic cells located in the basal layer of the epidermis, melanocytes. Melanogenesis takes place in organelles, the melanosomes, which, loaded with melanin, are transferred to the neighboring epidermal cells, the keratinocytes, via the dendrites.
This pigmentation can be modulated by physical factors, such as UV radiation, or chemical factors, such as depigmenting or propigmenting products, and/or during physiological or pathological modifications of the functioning of the skin.
It is therefore desirable to have models which, on the one hand, make it possible to carry out the studies necessary for a better understanding of the role of the skin, both in mechanical terms and in physiological terms, and which, on the other hand, constitute predictive tests for the activity of cosmetic and/or pharmaceutical active agents or else for the side effects of topical ingredients or agents taken orally.
At the current time, in vitro models exist for evaluating a modulation of pigmentation. They are in particular the following models:
inhibition, in tubo, of recombinant human or fungal tyrosinase (solely biochemical, non-cellular model) (Virador Analytical Biochem., 1999);
monolayer monocultures of normal melanocytes or melanocytes derived from melanoma (Virador Analytical Biochem., 1999, Ni-komatsu JID 2007);
monolayer co-culture of normal or immortalized human melanocytes-keratinocytes (Régnier Cell Mol Biol., 1999, Yoon Pigment Cell Res 2003);
pigmented reconstructed epidermis on dead de-epidermalized dermis or on an inert polycarbonate filter: 3D model constituted of melanocytes and keratinocytes only.
For many years, efforts have been made to develop reconstructed skin models which, on the one hand, make it possible to perform the studies necessary for a better understanding of the role of the skin, both in mechanical terms and in physiological terms, and which, on the other hand, constitute predictive tests for the activity of cosmetic and/or pharmaceutical active agents or else for side effects of topical ingredients.
EP 285471 thus describes a method for preparing a skin equivalent from a culture of keratinocytes originating from the sheath of the hair follicle; by seeding on a dermis equivalent, a differentiated epidermis, the structure of which is close to that of the human epidermis, is obtained.
Various skin equivalents have subsequently been proposed, and it has been suggested to introduce melanocytes into these models.
FR 2 689904 and WO 95/10600 describe the preparation of an epidermis equivalent containing keratinocytes and melanocytes, and the use thereof in sun-tanning tests. However, these epidermis equivalents are obtained on an inert support which does not reproduce the physiological interactions with the dermis, and which does not contain fibroblasts or a matrix environment. Furthermore, the culture conditions do not make it possible to keep the melanocytes in a normal phenotype owing to the use of a tumor promoter (TPA or PMA).
However, none of these models makes it possible to provide an understanding of the modulation of pigmentation by the participants of the dermis (fibroblasts, extracellular matrix, dermo-epidermal junction components) since they do not comprise a live dermis.
Even when human organotypic models comprising live fibroblasts are developed, they do not make it possible to evaluate a modulation of pigmentation in a physiological context because they have functionality deficiencies (absence of constitutive pigmentation or pigmentation that can be induced in particular by UV exposure).
Models of pigmented skin have been developed on de-epidermalized dead dermis or on sponge (matrix of collagen+GAGs) recolonized by fibroblasts. However, the localization of the melanocytes therein is often imperfect and these models exhibit functionality deficiencies. Specifically, they show little or no constitutive pigmentation, and the stimulation of pigmentation by UV radiation or by a physiologically propigmenting agent is not observed in these models.
Fibroblasts embedded in a matrix of free collagen (not taut) have also been used to reconstruct a pigmented skin. However, these models have drawbacks which do not enable them to be used to evaluate the modulation of pigmentation.
Bertaux et al. (Br J Dermatol., 1988) developed a model of pigmented skin by introducing a skin biopsy onto the dermal equivalent in order for the epidermal cells to leave the epidermis and proliferate on the surface of the lattice. Among the drawbacks of such a technique, exemplary is the lack of control of the epidermal cells and the fact that only one skin reconstruction is possible with a biopsy: no experiment is therefore comparable to the other (no reproducibility). Furthermore, like the model developed by Haake and Scott (Scott and Haake, J. Invest Dermatol., 1991), this model does not show any real functionality: neither the constitutive nor the induced presence of melanin is observed.
In the model by Archambault et al. (J Invest Dermatol., 1995), melanogenesis stimulation is induced after UV-B radiation, but for doses that induce epidermal alteration (cytotoxic dose) constituting conditions that are completely unacceptable for being able to study pigmentation and the modulation thereof in an in vitro context close to the physiological context.
Other more recent attempts at reconstructing a pigmented reconstructed skin have been made on a free lattice support; however, the models obtained do not correspond to models of normal human skin because, either they are created using mouse melanocytes (Yoshimura J Dermatol Sci., 2001), or the melanocytes are cultured under conditions which induce tumor transformation, in the presence of TPA (Liu Cell Biol Int., 2007). As for the model developed by Martinhao Sauto et al. (Sao Paulo Med J., 2006), no melanocyte is present or it is not shown in the epidermis.
Need therefore exists for a model making it possible to obtain a relevant understanding of the skin and the disruption thereof, reproducing the physiological interactions of human skin.
Need also exists for methods for evaluating the modulation of skin pigmentation, which make it possible to gain a relevant understanding of all the mechanisms involved in skin pigmentation and the disruption thereof, reproducing the physiological interactions of human skin.
In particular, need exists for a skin equivalent comprising functional live melanocytes having a similar localization to that which exists in human skin and a dermal compartment with live fibroblasts.